Mitogen-activated protein kinases (MAPKs) are universal elements of signaling pathways. ERK2 has long served as a prototype to define properties of MAPKs and their cascades. ERK2 and/or ERK1 are required not only for embryogenesis, differentiation, and proliferation, but also for events in differentiated cells, e.g., changes in membrane permeability, long term potentiation, and transcription. The misregulated or otherwise inappropriate functions of ERK1/2 contribute to diseases ranging from cardio-facio-cutaneous syndrome to polycystic kidney disease to cancer. We propose studies to discover how ERK1/2, essential, ubiquitous signaling proteins, act with specificity in different signal transduction processes. We will define ligand-dependent regulatory mechanisms that direct ERK1/2 to sites of action and determine functions at these sites. An essential site of action of ERK1/2 is the nucleus. The stimulus-induced nuclear translocation of phosphorylated ERK1/2 is required for several transcriptional responses and changes in cell programs. We will elucidate the underlying mechanisms of the nuclear import and export of ERK1/2. We have demonstrated that the nuclear translocation of phosphorylated ERK1/2 occurs only in response to a subset of ligands. We will examine how this specificity is achieved. We have also found that ligands stimulate the association of ERK1/2 with specific genes. We will determine how this occurs, how it is regulated, and the ligand-specific actions of ERK1/2 at these sites. Association of ERK1/2 with specific complexes controls their ligand-selective activation and localization to sites of action outside the nucleus as well. We will determine how cAMP differentially regulates the ERK1/2 pathway in response to growth factors. We will analyze complexes with cytoskeletal proteins and proteins we identified in a screen for interactors that selectively recognize phosphorylated ERK2. The structural basis for the interactions, the substrates and actions of the kinases in complex with these proteins, and the effects of these proteins on localization of ERK1/2 will be examined. The goals are to identify ligand-specific outputs and to have access to ERK1/2 substrates in the context of a specific output. Finally, the actions of calcineurin (PP2B) and phosphoprotein phosphatase 2A (PP2A) in ligand-specific regulation and function of ERK1/2 will be studied. The experiments proposed will determine mechanisms underlying ligand/process specificity in the ERK1/2 pathway.